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INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA

Peripheral Phosphodiesterase 4 Inhibition Produced by 4-[2-(3,4-Bis-difluoromethoxyphenyl)-2-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-phenyl]-ethyl]-3-methylpyridine-1-oxide (L-826,141) Prevents Experimental Autoimmune Encephalomyelitis

Departments of Pharmacology (C.S.M., N.E., A.L.O.H., G.S.R.) and Psychiatry (G.S.R.), Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada; Departments of Biology and Medicinal Chemistry, Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Quebec, Canada (R.F., A.S., J.A.M., D.W.N.); and Neuroimmunology Unit, Montreal Neurological Institute, Montreal, Quebec, Canada (T.O.)

Received for publication April 12, 2006
Accepted June 28, 2006.

	Abstract

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Administration of phosphodiesterase 4 (PDE4) inhibitors suppresses the pathogenesis associated with experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). In the present study, we compared the effects of rolipram and 4-[2-(3,4-bis-difluoromethoxyphenyl)-2-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-phenyl]-ethyl]-3-methylpyridine-1-oxide (L-826,141), a novel nonbrain penetrant PDE4 inhibitor, on the onset and severity of clinical signs in a chronic, nonrelapsing/remitting model of EAE. Both rolipram (10 mg/kg p.o.) and L-826,141 (3 mg/kg p.o.) reduced the severity of EAE relative to controls, whereas L-826,141 (3 mg/kg p.o.) also delayed disease onset. To assess whether L-826,141 prevented EAE progression after the first signs of clinical onset, rolipram (10 mg/kg p.o.) or L-826,141 (3 or 30 mg/kg p.o.) were administered 24 h after the first signs of EAE were observed. Only L-826,141 at a dose of 30 mg/kg p.o. significantly decreased the clinical severity of EAE compared with vehicle controls. Immunohistochemical detection of the neuronal activity marker Fos confirmed that L-826,141 did not reach concentrations in the central nervous system sufficient to activate central neurons. Lipopolysaccharide-induced tumor necrosis factor- in whole blood and plasma concentrations of L-826,141 revealed that only the 30-mg/kg dose resulted in levels sufficient to produce a near complete inhibition of PDE4 activity in immune cells. Taken together, these results demonstrate that peripheral PDE4 inhibition, produced by L-826,141, prevents the progression of EAE after the first onset of clinical signs, and suggest that similar compounds may have clinical efficacy in the treatment of MS.

Experimental autoimmune encephalomyelitis (EAE) is a predominantly Th1 cell-mediated autoimmune disease that is used as a model of MS in both rodents and nonhuman primates. Traditionally, EAE is induced by immunizing animals with specific myelin antigens, resulting in an immune response against the recognized antigen. The combination of myelin antigen and animal species/strain often predicts the clinical course of EAE, albeit chronic, acute, or relapsing-remitting (von Budingen et al., 2002). The pathological features of EAE are similar to those that are observed in MS, including blood-brain barrier breakdown, infiltration of immune cells in the CNS, inflammation, and demyelination (Floris et al., 2004; Pomeroy et al., 2005).

Phosphodiesterases (PDEs) are enzymes responsible for regulating levels of cyclic nucleotides, in particular cAMP and cGMP, which are important secondary messengers involved in molecular signaling. In particular, PDE4 is a cAMP-specific isoenzyme family of PDEs that is predominantly expressed in many inflammatory cells (Schudt et al., 1995). In immune cells, elevations in cAMP reduce cell motility (Layseca-Espinosa et al., 2003) and attenuate immune cell activation (Souness et al., 2000). Previously, PDE4 inhibitors have been used for suppressing inflammation in animal models of arthritis, chronic obstructive pulmonary disease, heart disease, and MS (Burnouf and Pruniaux, 2002; Hamamoto et al., 2004). The anti-inflammatory properties of PDE4 inhibitors have also led investigators to assess their therapeutic potential in a variety of human inflammatory and autoimmune diseases, including rheumatoid arthritis (Tenor et al., 2002) and asthma (Compton et al., 2001).

In immune cells, inhibition of PDE4 prevents cAMP degradation, thus elevating cAMP levels and inhibiting the production and release of proinflammatory cytokines from activated peripheral mononuclear cells, including but not limited to, tumor necrosis factor (TNF)-, interleukin (IL)-2, IL-12, and interferon (IFN)- (Kaminuma et al., 1998; Muise et al., 2002; Claveau et al., 2004). Consequently, TNF- concentrations have been used as a surrogate marker for examining inhibition of PDE4 activity (Muise et al., 2002). More recently, the enzymes encoded by the PDE4 splice variants PDE4A, PDE4B, and PDE4D are thought to be largely responsible for total PDE4 activity (Giembycz et al., 1996). In particular, PDE4B induction has been shown to be essential for LPS-activated TNF- responses in vivo (Jin and Conti, 2002). In terms of selectivity, L-826,141 (for structure, see Claveau et al., 2004) is a highly selective PDE4 inhibitor that has been to shown to be at least 450-fold more potent at inhibiting PDE4 versus PDE1-11 (Claveau et al., 2004).

The prototypical PDE4 inhibitor rolipram, initially developed as an antidepressant, has potent anti-inflammatory properties in models of allergic and autoimmune disease, including EAE (Sommer et al., 1995; Jung et al., 1996). Rolipram readily crosses the blood-brain barrier, resulting in emetic side effects, which have hampered the clinical development of this compound (Robichaud et al., 2001). Newer PDE4 inhibitors, such as L-826,141, are significantly less brain penetrant and do not display emesis at doses that are anti-inflammatory (Frenette et al., 2002). In addition, ferrets fail to display emesis when orally administered 30 mg/kg L-826,141 (Frenette et al., 2002). In the rat, L-826,141 is well absorbed and possesses a bioavailability of 60% when delivered orally at 3 mg/kg. The anti-inflammatory effects of L-826,141 (1 mg/kg i.p.) have been demonstrated in a guinea pig asthma model of ovalbumin-induced bronchoconstriction and in a squirrel monkey model of antigen-induced bronchoconstriction (0.5-3 mg/kg) (Muise et al., 2002).

The purpose of the present study was to determine whether oral administration of L-826,141 was sufficient to delay and/or decrease the clinical symptoms associated with EAE. The doses of L-826,141 used in this experiment were chosen based on their ability to suppress LPS-induced TNF- release in mouse whole blood. Here we show that L-826,141 prevents the development of EAE when administered before and after the first clinical signs are apparent. Our results suggest that PDE4 activity in EAE was not mediated by central inhibition of PDE4 activity but rather in the periphery.

	Materials and Methods

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Reagents. The PDE4 inhibitor rolipram [4-[3-(cyclopentyloxy)-4-methoxy-phenyl]pyrrolid-2-one] was purchased from A.G. Scientific, Inc. (San Diego, CA). L-826,141 was synthesized and supplied by Merck Frosst (Montreal, QC, Canada). Both compounds were suspended in 1% methylcellulose and underwent sonication for 30 min at 4°C before administration.

LPS-induced TNF- Release Whole-Blood Assay. C57BL/6 mice were dosed orally with L-826,141 (3 and 30 mg/kg) and vehicle every 24 h for 3 days. At trough levels, whole blood was collected into microtainers containing lithium heparin (BD Biosciences, Franklin Lakes, NJ). LPS (serotype 0111:B4; Sigma-Aldrich, St. Louis, MO) derived from Escherichia coli (100 µg) was used to induce the release of TNF-, a surrogate marker for PDE4 activity in mouse whole blood. Freshly prepared LPS was prepared at a concentration of 5 mg/ml in 0.1% bovine serum albumin in phosphate-buffered saline and diluted to a final concentration of 100 µg of LPS/ml of blood. The blood was incubated for 4 h at 37°C in a humidified tissue culture incubator supplemented with 5% CO₂. Following this incubation, blood was centrifuged at 1400g for 10 min at 4°C, and the plasma was collected and stored at -80°C.

As a method for determining the optimal dose for administering L-826,141 to EAE-immunized mice, the suppression of LPS-induced TNF- release in whole blood was assessed using an ELISA and performed according to the manufacturer's protocol (BioSource International, Camarillo, CA). In brief, using a solid-phase sandwich ELISA and a streptavidin-peroxidase reaction, the intensity of the colored product is directly proportional to the amount of TNF- in the sample. The absorbance was read at 450 nm blanked against a chromagen blank, and the amount of TNF- in each sample was calculated using a recombinant mouse TNF-.

Determination of Plasma Concentrations of L-826,141. Mice were orally dosed every 24 h for three consecutive days, and plasma was collected 24 h after the last dose. Samples were stored at -80°C until they were analyzed by liquid chromatography-mass spectrometry (B9-atmospheric pressure chemical ionization) with an internal standard (Merck Frosst Centre for Therapeutic Research, Kirkland, QC, Canada).

Induction of EAE. EAE studies were performed on 6 to 8-week-old female C57BL/6 mice (Charles River Canada, Montreal, QC, Canada). Animals were housed with access to food and water ad libitum on a 12-h light/dark cycle. All protocols used in this experiment were conducted in accordance with approval by the University Committee on Laboratory Animals at Dalhousie University (Halifax, NS, Canada) in accordance with the guidelines set by the Canadian Council on Animal Care.

Mice were immunized with a 1:1 ratio of myelin oligodendrocyte glycoprotein (MOG) encephalitogenic peptide amino acids 35 to 55 (MOG_35-55) (Sheldon Biotechnology Centre, Montreal, QC, Canada) dissolved in 0.9% saline and Complete Freund's adjuvant (CFA) containing 0.5 mg of Mycobacterium tuberculosis H37RA (Difco; BD Diagnostics). On day 0, the MOG-CFA emulsion was administered subcutaneously on both sides at the base of the tail (100 µg/mouse). The additional immune adjuvant pertussis toxin (Sigma-Aldrich) in 1x Hanks' balanced salt solution was injected i.p. (200 ng/mouse) on the initial day of immunization and again on day 2. On day 7, the MOG + CFA immunization was repeated, with two subcutaneous injections into the upper flanks of each mouse.

Care and Clinical Evaluation of EAE Mice. The weights of the mice were recorded daily, and the clinical scores of the animals were assessed over the duration of 21 days. The following grading scheme was used to clinically score the animals: 0, no clinical signs; 0.5, hook tail; 1, flaccid/floppy tail; 2, beginning of walking deficits; 3, unilateral hindlimb paralysis; 4, bilateral hindlimb paralysis; and 5, moribund. Clinical scoring was performed by two qualified individuals, one individual who was unaware of the treatment group. No significant differences were recorded between the recorded scores between the blinded and unblinded scorer. Mice were supplied with mash when they were no longer able to reach the food and water. Neutrical was also provided to mice as an additional nutrient supplement.

Rolipram and L-826,141 Administration. Rolipram (10 mg/kg), L-826,141 (3 mg/kg), or 1% methylcellulose (10 ml/kg) was used as the vehicle control and was administered daily by oral gavage beginning on day 9 (n = 12 mice/group). A second control group of immunized animals (n = 12) was not gavaged to assess the impact of gavage-induced stress on EAE severity. The two treatment groups receiving the PDE4 inhibitors were administered the compounds daily, for 12 days, until day 21, at which point the animals were sacrificed. To assess the effect of L-826,141 on decreasing EAE severity after the onset of clinical signs, mice were administered L-826,141 (3 mg/kg p.o.; n = 24), rolipram (10 mg/kg p.o.; n = 24), or vehicle (n = 16) 24 h after the onset of clinical signs of EAE, and they were dosed daily for the duration of the experiment. In this experiment, all mice were immunized on day 0; however, drug/vehicle treatment did not begin until each individualized mouse began to develop signs of EAE; thus, initiation of compound administration occurred over a period of days.

To determine whether an increased dose of L-826,141 had an impact on EAE severity, mice were either administered L-826,141 (30 mg/kg p.o.; n = 8) or vehicle (n = 11) 24 h after the onset of clinical signs of EAE and dosed daily for the duration of the experiment

Histology. To examine for the presence of cellular infiltrates and perivascular cuffing, an H&E stain was performed. Mice were deeply anesthetized using sodium pentobarbital and transcardially perfused using 10 ml of saline and fixed using 4% PFA in PB. The brains and spinal cords of animals were removed and postfixed in 4% PFA and stored at 4°C. Before sectioning brains and spinal cords, the tissues were cryoprotected in 30% sucrose in PB. Sagittal spinal cord sections (14 µm) were cut on the cryostat and stained with Mayer's hematoxylin and eosin (Sigma-Aldrich). In addition, sections were stained with FluoroMyelin Green fluorescent myelin stain and DAPI nucleic acid stain (Invitrogen, Carlsbad, CA) for assessment of demyelination and cellular infiltration.

c-fos and Glial Fibrillary Acidic Protein Immunohistochemistry. Brains and spinal cords were dissected from mice that received 3 mg/kg L-826,141, 10 mg/kg rolipram, or vehicle. Following a postfixation in 4% PFA and cryoprotected in 30% sucrose solution in PB, 30-µm sections of brains were cut on a freezing microtome. Brain sections were incubated for 30 min in a 1% H₂O₂ solution to eliminate endogenous peroxidase activity. The tissue was then blocked in 10% normal goat serum for 1 h at room temperature and then incubated with a polyclonal rabbit anti-mouse c-fos antibody (1: 5000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4°C. Tissue sections were rinsed with 0.3% PBS-Triton X-100 and then incubated with a biotinylated goat anti-rabbit secondary for 1 h at room temperature (1:500; Vector Laboratories, Burlington, ON, Canada). The tissue was subsequently exposed to an avidin-biotin-peroxidase complex (ABC kit; Vector Laboratories) for 1 h and developed using a glucose oxidase-stimulated diaminobenzidine reaction.

For examination of GFAP, spinal cord sections from mice that received either L-826,141 (3 mg/kg p.o.) or vehicle beginning on day 9 were used. Spinal cord sections (14 µm) were cut on the cryostat and incubated in a Cy-3 conjugated monoclonal antibody against glial fibrillary acidic protein antibody (1:1000) (Sigma-Aldrich) overnight at 4°C.

Data Analysis and Statistics. The effect of varying doses of L-826,141 on TNF- concentrations was analyzed using a one-way analysis of variance (ANOVA) for independent groups corresponding to different drug concentrations (three) Tukey's honestly significant multiple comparisons were used, and the 0.05 level of significance was adopted for all comparisons.

The effect of varying doses of L-826,141 on blood plasma concentrations was analyzed using a one-way ANOVA for independent groups, corresponding to different drug concentrations (three). Tukey's honestly significant multiple comparisons were used, and the 0.05 level of significance was adopted for all comparisons.

Clinical scores and corresponding weights for mice were subjected to separate one-way ANOVA for independent groups corresponding to different drug treatments (L-826,141) with repeated measures over days 0, 2, and 7 to 21. The overall ANOVA was followed by analyses of the simple main effects of drug treatment performed on independent days. Tukey's honestly significant multiple comparisons were used where appropriate, and the 0.05 level of significance was adopted for all comparisons.

	Results

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Plasma Concentrations of L-826,141 in C57BL/6 Mice Dosed Orally at 3 and 30 mg/kg. Three groups of mice were dosed with either vehicle, L-826,141 (3 mg/kg p.o.), or L-826,141 (30 mg/kg p.o.) every 24 h for 3 days. Using mass spectrometry, plasma levels of L-826,141 were determined 24 h after the last dose. Mice dosed with L-826,141 (3 mg/kg p.o.) had an average plasma concentration of 0.035 ± 0.007 µM (Fig. 1A). The average plasma concentration of L-826,141 in mice dosed at 30 mg/kg p.o. was 0.81 ± 0.08 µM. As expected, L-826,141 was not detectable in mice dosed with vehicle.

View larger version (17K): [in this window] [in a new window]   Fig. 1. A, plasma concentrations were obtained from mice dosed with L-826,141 (3 or 30 mg/kg p.o.) or vehicle. Three groups of mice were orally dosed with either vehicle, L-826,141 (3 mg/kg p.o.), or L-826,141 (30 mg/kg p.o.) every 24 h for 3 days, and plasma was collected. Mice dosed with L-826,141 (3 mg/kg p.o.) had an average plasma concentration of 0.035 ± 0.007 µM. The average plasma concentration of L-826,141 in mice dosed at 30 mg/kg p.o. was 0.81 ± 0.08 µM. Mice dosed with vehicle had a plasma concentration of 0 mg/kg. ***, significantly different from 1% methylcellulose group at p < 0.001. , significantly different from L-826,141 (3 mg/kg p.o.) group at p < 0.001. B, TNF- concentration in plasma of whole blood incubated with LPS, measured by an ELISA. Three groups of mice were administered either 1% methylcellulose (n = 3), L-826,141(3 mg/kg; n = 4), or L-826,141 (30 mg/kg; n = 3) once daily for 3 days. Blood was collected 24 h after the final administration of either drug or vehicle, and LPS-induced TNF- release was measured using a mouse TNF- ELISA (BioSource International). Data are expressed as the mean ± S.E.M. *, significantly different from vehicle group at p < 0.05; , significantly different from 3-mg/kg group at p < 0.01.

Oral Administration of L-826,141 (3 and 30 mg/kg) Significantly Inhibits LPS-Induced TNF- Release in Whole Blood from C57BL/6 Mice.

Oral Administration of L-826,141 (3 mg/kg) Prevents Weight Loss, Delays Onset, and Decreases Severity of EAE When Administered before the Onset of the Clinical Signs of EAE. Weight loss, onset, and severity of EAE were assessed in mice treated with L-826,141 (3 mg/kg p.o.), rolipram (10 mg/kg p.o.), or 1% methylcellulose (10 ml/kg p.o.), before the onset of clinical signs of EAE. To control for the effects of gavaging on EAE, an additional group of mice was immunized and did not receive either drug or vehicle. The nongavaged and 1% methylcellulose control groups displayed no weight gain over the duration of the experiment (Fig. 2A). In contrast, mice administered L-826,141 (3 mg/kg p.o.) displayed an increase in body weight compared with vehicle-treated mice. Rolipram-treated animals (10 mg/kg p.o.) displayed a similar trend in weight gain as the L-826,141-treated group. [drug, F(3,44) = 0.939, p = 0.43; day, F(15,660) = 1.755, p = 0.192; and day x drug, F(45,660) = 5.271, p = 0.003].

View larger version (33K): [in this window] [in a new window]   Fig. 2. Effects of orally administered PDE4 inhibitors rolipram (10 mg/kg) and L-826,141 (3 mg/kg) on the average weight (A) and clinical scores (B) of EAE-induced C57BL/6 mice. Four groups of mice were induced with EAE on day 0 (n = 12 mice/group) and vehicle (1% methylcellulose), or drug treatments were administered daily from day 9 to 20. An additional group of EAE mice was not gavaged. Data are expressed as the mean ± S.E.M. *, significantly different from 1% methylcellulose group at p < 0.05.

Mice administered L-826,141 (3 mg/kg p.o.) before the onset of EAE symptoms did not display clinical signs until day 17 (Fig. 2B). By contrast, mice treated with rolipram (10 mg/kg p.o.) and vehicle began to present clinical signs on day 10. The average clinical score of mice pretreated with L-826,141 (3 mg/kg p.o.) or rolipram (10 mg/kg p.o.) was lower compared with the control groups throughout the duration of the experiment. Over the 3-week period, mice treated with either L-826,141 or rolipram showed similar clinical scores. In addition, the 1% methylcellulose and nongavaged control groups also showed similar clinical scores throughout the experiment, suggesting that oral gavage does not influence the onset or development of EAE. Following EAE induction, the incidence of EAE was 17% in the group of mice pretreated with L-826,141 (3 mg/kg p.o.) compared with 25, 67, and 75% of mice in the rolipram, vehicle, and nongavaged control groups, respectively. Compared with the rolipram and control groups, the onset of EAE was also delayed in mice receiving L-826,141 (3 mg/kg p.o.) [drug, F(3,44) = 6.503 p < 0.001; day, F(15,660) = 36.442, p < 0.001; and day x drug, F(45,660) = 5.609, p = 0.002].

Oral Administration of L-826,141 (3 mg/kg) or Rolipram (10 mg/kg) Does Not Influence EAE Severity When Administered 24 h after the First Clinical Signs of EAE. In a second set of experiments, three groups of mice immunized with MOG_35-55 were permitted to develop clinical signs of EAE before administration of L-826,141, rolipram, or vehicle. Rolipram (10 mg/kg p.o.), L-826,141 (3 mg/kg p.o.), or vehicle was administered 24 h after the first appearance of clinical signs of EAE. Throughout the duration of the experiment, no differences in body weights were observed between the three treatment groups (Fig. 3A) [drug, F(2,60) = 2.393, p = 0.1; day, F(15,915) = 0.018, p = 0.893; day x drug, F(30,900) = 0.420, p = 0.659]. In addition, no differences in EAE severity were observed between the treatment groups (Fig. 3B). Beginning on day 19, mice treated with L-826,141 (3 mg/kg p.o.) showed a nonsignificant trend for reduced EAE severity, suggesting L-826,141 had a delayed degree of effectiveness when administered after the onset of EAE. By contrast, mice treated with rolipram or vehicle did not seem to show this trend after the onset of dosing [drug, F(2,60) = 1.599, p = 0.211; day, F(15,900) = 72.004, p < 0.001; day x drug, F(30,900) = 1.080, p = 0.346].

View larger version (29K): [in this window] [in a new window]   Fig. 3. Effects of orally administered PDE4 inhibitors rolipram (10 mg/kg p.o.) and L-826,141 (3 mg/kg p.o.) on the average weight (A) and clinical scores (B) of EAE-induced C57BL/6 mice 24 h after the onset of the first clinical signs of EAE. Three groups of mice were induced with EAE on day 0 and administered rolipram (10 mg/kg p.o.; n = 24), L-826,141 (3 mg/kg p.o.; n = 24), or vehicle (1% methylcellulose; n = 16). Data are expressed as the mean ± S.E.M.

EAE Experiment 3. Oral Administration of L-826,141 (30 mg/kg p.o.) Suppresses Weight Loss and Decreases EAE Severity When Administered 24 h after the Onset of Clinical Signs of EAE. In a third experiment, mice immunized with MOG_35-55 were permitted to develop clinical signs of EAE before oral administration of either L-826,141 (30 mg/kg p.o.) or vehicle. A rolipram treatment group was omitted, because no obvious trend was observed when this PDE4 inhibitor was administered 24 h after the onset of clinical signs of EAE. Furthermore, signs of toxicity at the 10-mg/kg dose of rolipram prevented a further increase in dosage. Mice were administered L-826,141 (30 mg/kg p.o.) 24 h after the first clinical signs of EAE and dosed daily for the duration of the experiment. The average weight of mice treated with vehicle declined over the duration of the experiment. This trend was not observed in mice treated with L-826,141 (30 mg/kg p.o.), indicative of overall increased health in these mice (Fig. 4A) [drug, F(1,17) = 3.011, p = 0.101; day, F(15,255) = 4.120, p = 0.058; day x drug, F(15,255) = 8.155, p = 0.011].

View larger version (27K): [in this window] [in a new window]   Fig. 4. Effects of orally administered PDE4 inhibitor L-826,141 (30 mg/kg) on the average weight (A) and clinical scores (B) of EAE-induced C57BL/6 mice. Two groups of mice were induced with EAE on day 0 and administered L-826,141 (30 mg/kg p.o.) 24 h after the onset of the first clinical signs of EAE. Data are expressed as the mean ± S.E.M. *, significantly different from 1% methylcellulose group at p < 0.05.

Beginning on day 13, a notable difference in EAE severity was observed between the groups that received either L-826,141 (30 mg/kg p.o.) or vehicle (1% methylcellulose; 10 mg/kg p.o.). In contrast to the experiment where mice received 3 mg/kg p.o., mice that received 30 mg/kg p.o. did not develop clinical signs of EAE (Fig. 4B) [drug, F(1,17) = 22.140, p < 0.001; day, F(15,255) = 68.614, p < 0.001; day x drug, F(15,255) = 29.243, p < 0.001].

Histopathological Evaluation of L-826,141-Treated Mice. To assess the effects of L-826,141 on the histopathology of EAE, the spinal cords of vehicle and L-826,141-treated animals were stained with H&E. Histopathological evaluation was performed in mice from both the prophylactic and therapeutic dosing studies. Immune cell infiltration and perivascular cuffing were observed in the white matter of spinal cords in mice that received vehicle (Fig. 5, A and B). A marked absence of cellular infiltration was observed in animals administered L-826,141 (Fig. 5, C and D). Demyelination in the spinal cord was assessed using a FluoroMyelin stain. Demyelinated lesions were apparent in the white matter of vehicle-treated animals (Fig. 6A). These areas of demyelination were accompanied by immune cell infiltration, visualized by a nuclear DAPI stain (Fig. 6, B and C). A lack of demyelination and cellular infiltration was observed in mice treated with L-826,141 (Fig. 6, D-F). Regardless of the dosing regime of L-826,141, the presence of cellular infiltrates and demyelination was dependent on EAE clinical severity. Increased cellular infiltration and demyelination was observed in mice that were treated with L-826,141 (3 mg/kg p.o.) 24 h after disease onset. A notable decrease in EAE histopathology was observed in mice dosed prophylactically with L-826,141 (3 mg/kg p.o.) or therapeutically (30 mg/kg p.o.).

View larger version (139K): [in this window] [in a new window]   Fig. 5. Increased cellular infiltration and perivascular cuffing in the spinal cords of vehicle-treated EAE mice. Spinal cord sections were stained from vehicle-treated (A and B) and L-826,141 (3 mg/kg p.o.) (C and D)-treated mice and stained using H&E. Perivascular cuffing, a hallmark characteristic of MS and EAE, was observed in vehicle-treated EAE mice (A and B). Scale bar, 200 µm.

View larger version (92K): [in this window] [in a new window]   Fig. 6. Areas of demyelination are accompanied by increased cellular infiltration in the spinal cords of symptomatic EAE mice. Spinal cord sections from symptomatic EAE mice were stained with FluoroMyelin, a green fluorescent myelin stain (A) and nuclear counterstained using DAPI, which specifically binds to DNA (B). An overlay of the two images (C) illustrates increased cellular infiltration of immune cells in demyelinated lesions of areas in the spinal cord. Similar staining was performed on L-826,141-treated mice. Scale bar (D-F), 200 µm.

GFAP Immunoreactivity in the Spinal Cord of L-826,141-Treated Mice. Previous studies have noted a proliferation of astrocytes and reactive gliosis in the CNS of animals with EAE (Aquino et al., 1988). Astrogliosis was assessed in lumbar sections of the spinal cord using a monoclonal Cy3-conjugated GFAP antibody. A considerable amount of GFAP immunoreactivity, indicative of astrogliosis, was observed in the lumbar sections of vehicle-treated animals (Fig. 7A). A notable decrease in GFAP immunoreactive cells was apparent in spinal cord sections from mice pretreated with 3 mg/kg L-826,141 (Fig. 7B). Increased GFAP immunoreactivity was observed in mice were treated with L-826,141 (3 mg/kg p.o.) 24 h after disease onset. A notable decrease in GFAP was observed in mice dosed prophylactically with L-826,141 (3 mg/kg p.o.) or therapeutically (30 mg/kg p.o.).

View larger version (73K): [in this window] [in a new window]   Fig. 7. Distribution of GFAP-positive cells in the lumbar regions of spinal cords in mice treated with vehicle (A) and 3 mg/kg L-826,141 (B). Spinal cord sections were stained with a Cy-3 conjugated GFAP monoclonal antibody against GFAP and visualized using fluorescence microscopy. An increase in GFAP-positive cells, indicative of astrogliosis, was noted in the vehicle-treated symptomatic EAE mice compared with the asymptomatic PDE4-treated mice. Scale bar, 200 µm.

View larger version (74K): [in this window] [in a new window]   Fig. 8. Fos immunoreactivity in the cortex of mice dosed with vehicle (1% methylcellulose) (A), L-826,141 (3 mg/kg p.o.) (B), and rolipram (10 mg/kg p.o.) (C and D). D, high magnification of boxed in areas in C. Scale bar, 500 µm (A-C) and 200 µm (D). An increased level of c-fos immunoreactivity was noted in the somatosensory cortex of rolipram-treated mice compared with mice receiving either L-826,141 or vehicle.

	Discussion

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LPS-induced TNF- release was used to determine the dose of L-826,141 necessary to inhibit PDE4 activity in whole blood. Assays performed in vitro have previously determined that L-826,141 transcriptionally down-regulates TNF- in whole-blood assays with an IC₅₀ value of approximately 0.31 µM (Claveau et al., 2004). L-826,141 has also been shown to inhibit LPS-induced release of IL-12, granulocyte macrophage-colony-stimulating factor (GM-CSF), and IFN- (Claveau et al., 2004). Oral administration of L-826,141 (3 mg/kg p.o.) was sufficient to delay the onset and decrease EAE severity when administered before the first appearance of clinical signs; however, this dose did not significantly decrease clinical signs when administered 24 h after the first onset of clinical signs of EAE. When dosed at 3 mg/kg p.o., the plasma concentration of L-826,141 was approximately 0.035 µM at trough (24 h). The known IC₅₀ values of L-826,141 required to inhibit the release of peripheral blood cytokines TNF-, IL-2, GM-CSF, and IFN are approximately 0.88, 0.28, and 0.35 µM, respectively (Claveau et al., 2004). Mice dosed with L-826,141 (3 mg/kg p.o.) every 24 h for three consecutive days had a plasma concentration of L-826,141 below the IC₅₀ required to inhibit the release of proinflammatory cytokines that are known to contribute to the pathogenesis of EAE. In attempts to decrease EAE severity after clinical onset, a higher dose of L-826,141 (30 mg/kg p.o.) was administered. LPS-induced TNF- release whole-blood assays performed using whole-mouse blood from mice treated with L-826,141 (3 or 30 mg/kg p.o.) demonstrated a dose-dependent decrease in this measure of PDE4 activity. In contrast to the plasma concentrations of L-826,141 in the mice dosed with 3 mg/kg p.o., plasma concentrations achieved after dosing with 30 mg/kg p.o. were approximately 0.81 µM, a concentration that is greater than the IC₅₀ required to inhibit TNF-, GM-CSF, and IFN- release in LPS-induced mouse whole-blood assays. L-826,141 (30 mg/kg p.o.) administered daily 24 h after the first clinical signs of EAE prevented EAE progression. With the exception of rolipram, to the best of our knowledge, this is the first documentation of an orally administered nonbrain penetrant PDE4 inhibitor that prevents the clinical signs of EAE after disease onset. The average day of onset of EAE in animals that received prophylactic administration of L-826,141 (3 mg/kg p.o.) was significantly delayed compared with rolipram (10 mg/kg p.o.) and vehicle-treated animals. Using clinical scores and body weights as a method of assessing the general health of the animals, mice pretreated with L-826,141 seemed to remain healthy.

Nausea and emesis remain a large concern in the development of PDE4 inhibitors (Duplantier et al., 1996). Although rodents do not vomit, it is still possible to distinguish whether the mice are experiencing discomfort as a direct result of constant central PDE4 inhibition. We noted that immediately following administration of rolipram, mice showed visible signs of discomfort and lethargy (data not shown). In addition, rolipram-treated mice were aversively conditioned to the act of gavaging, suggestive of a learned negative association between gavaging and the central effects of rolipram. This behavior was not observed in mice treated with vehicle or L-826,141. In contrast, L-826,141-treated mice became more compliant as oral delivery of the drug was administered during the course of the experiment.

Rolipram possesses high affinity for its PDE4 binding sites in the brain, in particular the area postrema (Carpenter et al., 1988). In rodents, rolipram has previously been shown to increase Fos expression in the cerebral cortex (Svenningsson et al., 1995) caudate putamen, and nucleus accumbens (Thompson et al., 2004). In addition, other agents that affect intracellular cAMP, including forskolin and Ro 20-1724, a PDE4 inhibitor, also stimulate c-fos gene expression (Haas et al., 1991). Despite the ability of rolipram to suppress the clinical signs of EAE (Sommer et al., 1997), the therapeutic potential and clinical development of this compound is hindered by its high emetic potential (Burnouf and Pruniaux, 2002). To test whether central PDE4 inhibition was occurring in the brains off L-826,141-treated mice, levels of the putative neuronal activity marker Fos were assessed by immunohistochemistry 24 h after administration. Mice dosed with rolipram (10 mg/kg p.o.) showed increased Fos immunoreactivity in the somatosensory cortex and paraventricular regions of the thalamus, compared with vehicle controls. In L-826,141-treated mice, Fos immunoreactivity in the brain was comparable with that of vehicle-treated animals. Because the blood-brain barrier is compromised in EAE, it may be possible that L-826,141 could enter the CNS; however, we think this is unlikely for the following reasons: 1) animals treated prophylactically with L-826,141 did not develop EAE, indicating that peripheral PDE4 inhibition was sufficient to prevent the disease; 2) animals treated with rolipram, but not L-826,141, displayed signs of sedation following administration of this PDE4 inhibitor and seemed to find the drug increasingly aversive (resisted oral gavage) during the course of the study; and 3) a lack of Fos activation in emetic regions of the hindbrain following administration of L-826,141 (30 mg/kg p.o.) in EAE mice (data not shown). In addition, oral administration of L-826,141 (30 mg/kg p.o.) fails to produce emesis in ferrets (Frenette et al., 2002). Taken together, these data suggest that the anti-inflammatory actions produced by oral administration of L-826,141 occurred peripherally, reducing the concern of emetic side effects in the clinic.

Activation of the hypothalamic-pituitary-adrenal axis by stress results in the release of endogenous glucocorticoids that can alter susceptibility to certain autoimmune diseases (Morale et al., 2001). In the EAE model, restraint stress has been shown to have a significant impact on disease suppression in a murine model (Dowdell et al., 1999). In the present experiment, the influence of restraint stress by oral gavage on the suppression of EAE was investigated. Over a 12-day period, using both a gavaged 1% methylcellulose group and a nongavaged control, no significant differences in weight, disease onset, or clinical scores were recorded. It was therefore concluded that restraint stress associated with oral gavage did not influence the development and progression of EAE.

H&E and FluoroMyelin stains were performed on the spinal cords of EAE animals to assess lymphocyte infiltration and demyelination, respectively. Both of these hallmark features of EAE were observed in mice that possessed elevated clinical scores. As a method of assessing whether peripheral PDE4 inhibition prevented this pathology, the presence of demyelination and cellular infiltrates was examined in mice that were administered L-826,141. Asymptomatic mice that received L-826,141 (3 mg/kg p.o.) before the onset of clinical signs of EAE did not possess these histopathological features of EAE.

Following CNS trauma, inflammatory mediators, such as TNF-, often contribute to astrogliosis and elevated GFAP expression (Zhang et al., 2000; Kernie et al., 2001). Elevated GFAP expression is observed in the CNS of MS patients and may contribute to disease progression (Norgren et al., 2004). GFAP immunoreactivity was assessed in the spinal cords of EAE mice. Animals with elevated clinical scores showed an increase in GFAP immunoreactivity, indicative of astrogliosis, in lumbar regions of the spinal cord. At doses sufficient to attenuate or delay the onset of EAE, L-826,141 also decreased GFAP-positive immunoreactivity in the spinal cords of these animals.

In vitro, PDE4 inhibition suppresses T-cell proliferation and transendothelial migration and decreases production of IL-2 and -4, IFN-, and TNF- (Gantner et al., 1997; Pette et al., 1999). PDE4 inhibitors also suppress macrophage activation (Beshay et al., 2001) and TNF- release from monocytes (Seldon et al., 1995). The development of a drug that does not require entry into the CNS and also decreases T-cell activation, proliferation, and extravasation through the blood-brain barrier would be extremely beneficial to those individuals suffering from MS. The potency, low emetic potential, inability to accumulate in the CNS, and anti-inflammatory properties of L-826,141 make this compound a promising therapeutic candidate for MS.

In summary, oral administration of 3 mg/kg L-826,141 significantly delayed the onset and decreased the severity of EAE in C57BL/6 mice. After the initial symptoms of EAE were observed, a higher dose of L-826,141 (30 mg/kg p.o.) was required to significantly decrease EAE severity when administered 24 h after the onset of the first clinical signs of EAE. Unlike many PDE4 inhibitors that have been previously developed and tested in the EAE model, L-826,141 exerts its mechanism of action in the periphery and does not cause nausea and emesis. At doses that are anti-inflammatory, these results demonstrate that peripheral PDE4 inhibition is sufficient to suppress EAE pathogenesis, suggesting that compounds with properties similar to L-826,141 may be useful in the treatment of MS.

	Acknowledgements

 
We acknowledge Merck Frosst Centre for Therapeutic Research for the generation and donation of L-826,141. We also thank Lyne Bourbonniere for excellent technical support.

	Footnotes

 
G.S.R. holds a Canadian Institutes of Health Research Research-Based Pharmaceutical Companies Chair.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.106096.

ABBREVIATIONS: MS, multiple sclerosis; CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; PDE, phosphodiesterase 4; TNF, tumor necrosis factor; IL, interleukin; IFN, interferon; L-826,141, 2-(3,4-bis-difluoromethoxyphenyl)-2-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-phenyl]-ethyl]-3-methylpyridine-1-oxide; ELISA, enzyme-linked immunosorbent assay; MOG, myelin oligodendrocyte glycoprotein; CFA, Complete Freund's adjuvant; PFA, paraformaldehyde; DAPI, 4,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; ANOVA, analysis of variance; LPS, lipopolysaccharide; GM-CSF, granulocyte macrophage-colony-stimulating factor; PB, phosphate buffer; Ro 20-1724, 4-[(3-butoxy-4-methoxyphenyl)-methyl]-2-imidazolidinone.

Address correspondence to: Dr. George S. Robertson, Departments of Psychiatry and Pharmacology, Faculty of Medicine, Sir Charles Tupper Bldg., 5850 College St., Halifax, NS B3H 1X5, Canada. E-mail: george.robertson{at}dal.ca

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